ST8Sia IV (PST) is an 12,8-sialyltransferase that catalyzes the polysialylation of a limited number of substrate glycoproteins, with the neural cell adhesion molecule (NCAM) being the primary substrate. Polysialic acid addition to NCAM modulates cell adhesion by acting as an anti-adhesive that prevents cell interactions mediated by NCAM and other nearby adhesion molecules. Previous studies have demonstrated that polysialylated NCAM is absolutely necessary for proper development and plasticity of the mammalian nervous system and the regeneration of damaged neurons. Interestingly, polysialylated NCAM has been found to be aberrantly expressed in several human tumors, where it promotes their growth and invasiveness. Despite our knowledge of the important beneficial and detrimental effects of NCAM polysialylation, the mechanism by which PST recognizes and polysialylates NCAM is not well understood. Results from several laboratories lead to the hypothesis that polysialylation of NCAM by PST is a highly protein-specific process requiring an initial recognition step that is mediated through PST-NCAM protein-protein interactions. The overall goal of the proposed research is to further test this hypothesis and elucidate the mechanism of NCAM protein-specific polysialylation by identifying the amino acid sequences and residues within PST that are responsible for recognition and binding of NCAM. In Specific Aim I, competition, mutagenesis/reconstitution, and binding studies will be used to identify sequences in PST required for NCAM recognition. Preliminary results have demonstrated that a truncated, catalytically inactive PST protein consisting of amino acids 1-140 acts as a strong competitor of SW2 cell NCAM polysialylation. The ability of a series of further truncated catalytically inactive PST proteins to compete with endogenous PST in SW2 small cell lung carcinoma cells and block NCAM polysialylation will be evaluated using the anti-polysialic acid antibody, OL.28, immunoblotting and FACS analysis. PST sequences that function as effective competitors will further be tested for their ability to bind to NCAM using co-immunoprecipitation and isothermal titration calorimetry analyses. Those PST sequences that bind NCAM will be replaced in PST and inserted into a related 12,8-sialyltransferase, ST8Sia III, to determine whether they are necessary and sufficient for NCAM recognition leading to polysialylation. In Specific Aim II, structural analyses will be performed to identify amino acid residues within PST and those in NCAM that mediate the PST-NCAM interaction. The goal of this aim is to obtain a co-crystal structure of a PST peptide with the recently crystallized NCAM Ig5-FN1 fragment (minimal domain required for polysialylation by PST), and to initiate experiments directed at obtaining the crystal structure of soluble PST. Accomplishment of these aims will provide greater insight into the mechanism of NCAM polysialylation by PST and may serve as the basis for the development of therapeutic agents targeting the PST-NCAM interaction.